Altered glial glutamate transporter expression in descending circuitry and the emergence of pain chronicity.

BACKGROUND: The glutamate type 1 transporter (GLT1) plays a major role in glutamate homeostasis in the brain. Although alterations of GLT1 activity have been linked to persistent pain, the significance of these changes is poorly understood. Focusing on the rostral ventromedial medulla, a key site in pain modulation, we examined the expression and function of GLT1 and related transcription factor kappa B-motif binding phosphoprotein (KBBP) in rats after adjuvant-induced hind paw inflammation. RESULTS: After inflammation, GLT1 and KBBP showed an early upregulation and gradual transition to downregulation that lasted throughout the eight-week observation period. Nitration of GLT1 was reduced at 30 min and increased at eight weeks after inflammation, suggesting an initial increase and later decrease in transporter activity. Mechanical hyperalgesia and paw edema exhibited an initial developing phase with peak hyperalgesia at 4 to 24 h, a subsequent attenuating phase, followed by a late persistent phase that lasted for months. The downregulation of GLT1 occurred at a time when hyperalgesia transitioned into the persistent phase. In the rostral ventromedial medulla, pharmacological block with dihydrokainic acid and RNAi of GLT1 and KBBP increased nociception and overexpression of GLT1 reversed persistent hyperalgesia. Further, the initial upregulation of GLT1 and KBBP was blocked by local anesthetic block, and pretreatment with dihydrokainic acid facilitated the development of hyperalgesia. CONCLUSIONS: These results suggest that the initial increased GLT1 activity depends on injury input and serves to dampen the development of hyperalgesia. However, later downregulation of GLT1 fosters the net descending facilitation as injury persists, leading to the emergence of persistent pain.

Identification and characterization of a brilliant yellow pigment produced by Bordetella pertussis.

Culture supernatants of Bordetella pertussis are a brilliant yellow; however, the structure and biological role of the responsible pigment have not been investigated. In this study, a brilliant yellow-colored fraction was extracted from culture supernatants of B. pertussis and analyzed by HPLC. UV-visible spectral analysis and mass spectrometry identified the brilliant yellow pigment as riboflavin. Riboflavin production was high in lag and early log phases and riboflavin was found to enhance growth of B. pertussis in low-density cultures. Riboflavin production is not regulated by the BvgAS system. In addition, it was found that other Bordetella species, such as B. parapertussis, B. holmesii and B. bronchiseptica, also release riboflavin into their culture supernatants. This is the first report that B. pertussis secrets riboflavin to the extracellular space and that riboflavin may promote its growth. The mechanism may be associated with pathogenesis of B. pertussis.

Development of vaccines against pertussis caused by Bordetella holmesii using a mouse intranasal challenge model.

Bordetella holmesii is recognized as the third causative agent of pertussis (whooping cough) in addition to Bordetella pertussis and Bordetella parapertussis. Pertussis caused by B. holmesii is not rare around the world. However, to date, there is no effective vaccine against B. holmesii. We examined the protective potency of pertussis vaccines available in Japan and vaccines prepared from B. holmesii. A murine model of respiratory infection was exploited to evaluate protective potency. No Japanese commercial pertussis vaccines were effective against B. holmesii. In contrast, a wBH vaccine and an aBH vaccine prepared from B. holmesii were both protective. Passive immunization with sera from mice immunized with aBH vaccine established protection against B. holmesii, indicating that B. holmesii-specific serum antibodies might play an important role in protection. Immuno-proteomic analysis with sera from mice immunized with aBH vaccine revealed that the sera recognized a BipA-like protein of B. holmesii. An aBH vaccine prepared from a BipA-like protein-deficient mutant strain did not have a protective effect against B. holmesii. Taken together, our results suggest that the BipA-like protein plays an important role in the protective efficacy of aBH vaccine.

Enhancement and regulation effect of myrcene on antibody response in immunization with ovalbumin and Ag85B in mice.

BACKGROUND: MF59, which is an adjuvant belonging to C30 member of the terpene family, is a T helper type-2 (Th2)-biased immune enhancer. Our previous studies showed that pyriproxyfen, a member of the terpene family with fewer carbon atoms (C20) than MF59, enhanced active T helper type-1 (Th1)-biased immune responses. OBJECTIVE: This study was performed to investigate the enhancement of antigen-specific immune responses by myrcene, a member of the terpene family with fewer carbon atoms (C10) than pyriproxyfen. METHOD: Ovalbumin (OVA) was used as an antigen to determine the effects of myrcene on the immune response. The IgG subtypes and cytokines induced by immunization of OVA with or without myrcene were monitored. Thereafter, we determined the effects of myrcene in the immune response against Ag85B, which is a dominant protective antigen for tuberculosis. RESULTS: The results showed that 0.8 mg/dose of myrcene enhanced antigen-specific total IgG immune response to OVA. Direct mixing of the antigen with myrcene was required for the enhancement of antibody production. Myrcene increased OVA-specific IgG2a titer, suggesting induction of Th1-immune response. The level of Th1 cytokines, IFN-γ was increased at 8 weeks after immunization, although IL-13 was also increased at the same time point. However, finally myrcene was found to increase Ag85B-specific total IgG titers at 5 weeks and specific IgG2a titer was increased at both 5 and 8 weeks. The results suggested that myrcene could enhance Th1 immune response. CONCLUSIONS: Myrcene enhanced specific immune responses against OVA and Ag85B. This study suggested the tendency of the enhancement of Th1 immune response by myrcene.

Detection of antibodies against fimbria type 3 (Fim3) is useful diagnostic assay for pertussis.

Isolation of Bordetella pertussis and detection of the pertussis genome are not always successful because of low bacterial loads in adult patients with pertussis. Antibodies against pertussis toxin (PT) are measured but have low sensitivity in vaccinated subjects. There is no reliable diagnostic method at present. In this study, a fluorescent-EIA against several pertussis antigens and genome detection were investigated to establish clinical laboratory diagnostic methods for pertussis. The study was conducted in an outpatient clinic between September 2007 and 2013. Subjects consisted of 209 patients including adults suspected of pertussis and 35 staff members of the clinic. Loop-mediated isothermal amplification (LAMP) was performed to detect the pertussis genome in 5' UTR of the pertussis toxin (PT) gene. The catalytic region of the adenylate cyclase toxin (catACT), C-terminal of filamentous hemagglutinin (cFHA), and type 3 fimbria (Fim3) were selected, which are not pertussis vaccine component. Conventional PT and FHA antibodies were examined together with type 2 fimbria (Fim2) antibodies, and these are vaccine antigens. Pertussis DNA was detected in 23 (11%) out of 209. Detection sensitivity was high in young infants. Antibodies against Fim3 showed a higher positive rate in all age groups. Staff members at the pediatric outpatient clinic showed serological booster responses in Fim2 and Fim3 antibodies more sensitively than those in PT antibodies during outbreaks. LAMP was useful for detecting the pertussis genome in young infants, whereas a serological assay for fluorescent-EIA against Fim2 and Fim3 was preferable for adolescents and adults.

Pyriproxyfen enhances the immunoglobulin G immune response in mice.

Pyriproxyfen is a juvenile hormone mimic of vital importance for insect development with little risk to humans. This study was performed to investigate whether large doses of pyriproxyfen affect the immune response in mammals. Mice were immunized thrice with ovalbumin in 5% ethanol, with or without pyriproxyfen or alum. Large doses of pyriproxyfen (9 or 15 mM) significantly enhanced specific total IgG immune response. This enhancement was no longer present 24 hr after treatment with pyriproxyfen. These results suggest that pyriproxyfen is a safe chemical. Moreover, pyriproxyfen induced higher titers of IgG2a and enhanced tumor necrosis factor-alpha and gamma-interferon responses whereas alum induced IgG1 with enhanced interleukin-4 and -10. These observations indicate that the mechanism of immune enhancement by pyriproxyfen may differ from that of alum.

Bone marrow stromal cells produce long-term pain relief in rat models of persistent pain.

Chronic pain conditions are difficult to treat and are major health problems. Bone marrow stromal cells (BMSCs) have generated considerable interest as a candidate for cell-based therapy. BMSCs are readily accessible and are easy to isolate and expand ex vivo. Clinical studies show that direct injection of BMSCs does not produce unwanted side effects and is well tolerated and safe. Here, we show that a single systemic (intravenous) or local injection (into the lesion site) of rat primary BMSCs reversed pain hypersensitivity in rats after injury and that the effect lasted until the conclusion of the study at 22 weeks. The pain hypersensitivity was rekindled by naloxone hydrochloride, an opioid receptor antagonist that acts peripherally and centrally, when tested at 1-5 weeks after BMSC infusion. In contrast, naloxone methiodide, a peripherally acting opioid receptor antagonist, only rekindled hyperalgesia in the first 3 weeks of BMSC treatment. Focal downregulation of brainstem mu opioid receptors by RNA interference (RNAi) reversed the effect of BMSCs, when RNAi was introduced at 5- but not 1-week after BMSC transplantation. Thus, BMSCs produced long-term relief of pain and this effect involved activation of peripheral and central opioid receptors in distinct time domains. The findings prompt studies to elucidate the cellular mechanisms of the BMSC-induced pain relieving effect and translate these observations into clinical settings.

A novel multilocus variable-number tandem repeat analysis for Bordetella parapertussis.

Purpose. Human-adapted Bordetella parapertussis is one of the causative agents of whooping cough; however, there are currently no genotyping systems with high discriminatory power for this bacterial pathogen. We therefore aimed to develop a multilocus variable-number tandem repeat analysis (MLVA) for human-adapted B. parapertussis.Methodology. Four highly polymorphic variable number tandem repeat (VNTR) loci in the B. parapertussis genome were selected and amplified by multiplex PCR. MLVA was performed based on the number of tandem repeats at VNTR loci. The discriminatory power of MLVA was evaluated with three laboratory reference strains and 50 human isolates of B. parapertussis.Results. Multiplex PCR-based MLVA characterized 53 B. parapertussis reference strains and isolates into 25 MLVA types and the Simpson diversity index was 0.91 (95 % confidence interval, 0.86-0.97). The three reference strains exhibited different MLVA types. Thirty-one Japanese isolates, ten French isolates and three Taiwanese isolates belonged to fourteen, nine and three MLVA types, respectively. In contrast, all five Australian isolates belonged to the same type. Two Japanese isolates collected from patients with known epidemiological links had the same type.Conclusion. Our novel MLVA method has high discriminatory power for genotyping human B. parapertussis. Regarding this organism, this genotyping system is a promising tool for epidemiological surveillance and investigating outbreaks.

Glial-cytokine-neuronal interactions underlying the mechanisms of persistent pain.

The emerging literature implicates a role for glia/cytokines in persistent pain. However, the mechanisms by which these non-neural elements contribute to CNS activity-dependent plasticity and pain are unclear. Using a trigeminal model of inflammatory hyperalgesia, here we provide evidence that demonstrates a mechanism by which glia interact with neurons, leading to activity-dependent plasticity and hyperalgesia. In response to masseter inflammation, there was an upregulation of glial fibrillary acidic proteins (GFAPs), a marker of astroglia, and interleukin-1beta (IL-1beta), a prototype proinflammatory cytokine, in the region of the trigeminal nucleus specifically related to the processing of deep orofacial input. The activated astroglia exhibited hypertrophy and an increased level of connexin 43, an astroglial gap junction protein. The upregulated IL-1beta was selectively localized to astrocytes but not to microglia and neurons. Local anesthesia of the masseter nerve prevented the increase in GFAP and IL-1beta after inflammation, and substance P, a prototype neurotransmitter of primary afferents, induced similar increases in GFAP and IL-1beta, which was blocked by a nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester. Injection of IL-1 receptor antagonist and fluorocitrate, a glial inhibitor, attenuated hyperalgesia and NMDA receptor phosphorylation after inflammation. In vitro application of IL-1beta induced NR1 phosphorylation, which was blocked by an IL-1 receptor antagonist, a PKC inhibitor (chelerythrine), an IP3 receptor inhibitor (2-aminoethoxydiphenylborate), and inhibitors of phospholipase C [1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione] and phospholipase A2 (arachidonyltrifluoromethyl ketone). These findings provide evidence of astroglial activation by tissue injury, concomitant IL-1beta induction, and the coupling of NMDA receptor phosphorylation through IL-1 receptor signaling.

Lipooligosaccharide from Bordetella pertussis induces mature human monocyte-derived dendritic cells and drives a Th2 biased response.

Bordetella pertussis has a distinctive cell wall lipooligosaccharide (LOS) that is released from the bacterium during bacterial division and killing. LOS directly participates in host-bacterial interactions, in particular influencing the dendritic cells' (DC) immune regulatory ability. We analyze LOS mediated toll-like receptor (TLR) activation and dissect the role played by LOS on human monocyte-derived (MD)DC functions and polarization of the host T cell response. LOS activates TLR4-dependent signaling and induces mature MDDC able to secrete IL-10. LOS-matured MDDC enhance allogeneic presentation and skew T helper (Th) cell polarization towards a Th2 phenotype. LOS protects MDDC from undergoing apoptosis, prolonging their longevity and their functions. Compared to Escherichia coli lipopolysaccharide (LPS), the classical DC maturation stimulus, LOS was a less efficient inducer of TLR4 signaling, MDDC maturation, IL-10 secretion and allogeneic T cell proliferation and it was not able to induce IL-12p70 production in MDDC. However, the MDDC apoptosis protection exerted by LOS and LPS were comparable. In conclusion, LOS treated MDDC are able to perform antigen presentation in a context that promotes licensing of Th2 effectors. Considering these properties, the use of LOS in the formulation of acellular pertussis vaccines to potentiate protective and adjuvant capacity should be taken into consideration.